It is frequently desirable in medical laboratory practice as well as in biological research to grow cells or tissues in particular media and then to examine the resulting growth. This procedure can be carried out by growing cells in one media and then transferring the cells to a microscope slide for optical examination. For example, cells can be grown in tissue culture flasks or bottles or in multi-well (micro-titer) test plates. These multi-well plates are well known and exemplified in U.S. Pat. Nos. 3,540,857 and 3,540,858.
Microtiter plates can be injection molded from polystyrene. Typically, such test plates have been standardized in forms that provide ninety-six depressions or cylindrical wells, each being about 0.66 cm in diameter and about 1.3 cm deep, arranged in a 12.times.8 regular rectangular array with the wells being spaced about 0.9 cm center to center. A flat lid is employed for covering the plates during incubation. Cell cultures can be incubated in the wells and in-vitro assays can be performed on the cultures.
Microtiter plates offer an advantage over flasks and bottles by allowing for the simultaneous growth of individual cultures and assays. None of these devices, however, provides a flat clear viewing surface for fluorescence and confocal imaging of cells. Therefore, cells from a produced culture are transferred to a glass microscope slide or coverslip for optical viewing. This procedure of growing a culture in one container and transferring the culture or cells therefrom for microscopic viewing has its downfalls in that the cells thus observed are not in their native state. Thus, true in-situ observation does not occur. The transfer procedure also requires an additional manipulative step.
U.S. Pat. No. 3,726,764 to White discloses a chamber attached to a glass slide with a liquid-impermeable seal. A special tool is wedged into the seal to separate the chamber from the glass slide. A problem which has confronted users of typical apparatus as described in U.S. Pat. No. 3,726,764 is that a separate tool to remove the chamber from the slide is not convenient and in spite of the high level of skill and care in separating the chamber and the slide, the potential for not shattering the glass slide is not always assured, and therefore, contamination of the culture on the slide is not assured. Furthermore, although the receptacle may be sealed to the base member, the patent does not disclose a completely liquid-impermeable assembly as there is no provision for sealing the lid 18 onto the assembly.
U.S. Pat. No. 3,745,091 to McCormick discloses an assembly comprising a microscope slide as a base member providing a planar surface and a receptacle formed of upstanding sidewalls and upstanding endwalls. The receptacle is attached to the base by a removable adhesive gasket. Partitions form a unitary structure with the sidewalls and endwalls and define cubical chambers in the receptacle. The receptacle, which can form one to eight cubical chambers, is preferably formed from a transparent organoplastic material, such as polystyrene, polypropylene, polymethacrylate, and the like. The adhesive gasket is made of, for example, an organopolysiloxane elastomer. A cover is used to seal the device. Cells and liquid media are placed in the cubicals, covered, and incubated. The liquid media is removed from the chamber and the receptacle removed from the base. The cell culture growth on the base glass is then treated as desired and examined microscopically. Unfortunately, this device has many disadvantages, for example, the gasket is often difficult to remove resulting in breakage of the glass slide, and the size of the cubicals are relatively large requiring a minimum of 100 .mu.l liquid. This type of device is currently sold as Lab-Tek Chambered Coverglass Products by Nunc, Inc, Naperville, Ill. Use of this type of device is described more fully by Simpson et al. (1985), Tsai et al. (1992), and Piazza et al. (1994).
Current molecular biology technology which now allows for the detection of a single copy of a specific gene or gene sequence has permitted the use of smaller and smaller numbers of cells for conducting biological research. Thus the number of cells required for a variety of biochemical assays, such as cell growth and attachment studies, cell differentiation studies, in-situ hybridization, and immunohistochemical procedures, has been greatly reduced. The reduction in assay size for these biological assays leads to a reduction in the use of expensive reagents and offers the opportunity to conduct more assays simultaneously.
Glass microscope slides with one or more reaction fields which are bounded by a hydrophobic surface coating are used for a wide variety of biological assays. These devices which are available from Cell-Line Associates, Newfield, N.J., Erie Scientific, Rye, N.H. and Precision Scientific, Madison, Wis., and are described in U.S. Pat. No. 4,705,705 to Bross. Typically, these glass slides have 5-10 mm wells which can hold from 20 to 100 .mu.l of fluid. The wells contain considerably less fluid than the Lab-Tek or Super Cell devices thus producing a considerable savings in cost of reagents. In addition, the reduction in size allows many more wells to be tested simultaneously. Slides can easily contain 16-64 wells. Cells can be transferred from cell culture devices to the wells of the slide for assaying and microscopic viewing. Cells can either be in suspension or fixed to the surface of the slide. Subsequently, reagents may be added to the wells and held in place by forces of surface tension. These slides are used for numerous types of investigations, namely:
1. morphological investigations of cells following fixing and staining, in a manner similar to the normal smear techniques and cytocentrifuging;
2. incubation of cells with various antibodies against cell membrane antigens for the identification of specific cell populations, with subsequent visualization of reaction by means of antibody labeling with enzymes, fluorescent dyes or gold particles;
3. detection of intracellular antigens by means of labeled antibodies, following drying and fixing of the cells;
4. detection by reactions of the cells with particles such as bacteria, latex particles, acrylic particles etc., and with substances such as dyes, toxins, and lectins;
5. performance of cytochemical reactions for the detection of cellular enzymes; and
6. coating and processing of tissue sections for pathological examination.
For investigations (1)-(5) above it would be a considerable advantage to grow cells directly on these devices in order to perform true in-situ assays on small number of cells and to eliminate the step of transferring cells from a different growth environment, the solution to which problem the present invention addresses. All of the above investigations (1)-(6) may have single or multiple incubations steps.
A recent modification of the Lab-Tek device replaces the aforementioned gasket with an acrylic pressure sensitive adhesive, as described in U.S. Pat. No. 5,571,721 to Turner, which discloses a slide and a cover adhered to the slide with the acrylic adhesive. According to the patent, substantially all of the acrylic adhesive remains attached to the cover when the cover is removed from the slide. A common problem which has confronted users of apparatus as described in U.S. Pat. No. 5,571,721 is that the seal provided by the acrylic adhesive between the slide and the cover is neither leak-proof, liquid impermeable, nor resealable. Furthermore, because the adhesive is removed with the cover, if a more permanently storable container is to be provided, as, for example, when a coverslip is placed over the culture and glued to the slide along the periphery of the coverslip, additional adhesive must be delicately placed on the slide, carefully surrounding the culture. Furthermore, a special device is needed to remove the receptacle and pressure sensitive adhesive from the glass slide, and removal of the receptacle still results in occasional breakage of slides. After separation from the slide, the receptacle cannot be resealed to the slide to form a leak-proof seal. Devices of this type are known as Super Cell Culture Slides, manufactured by Erie Scientific, Rye, N.H., and are sold with 1 to 16 individual chambers.
In addition to the aforementioned problems, none of the foregoing mentioned assemblies provides a protective cover sealed to a base wherein the cover can be temporarily removed from the base to gain access to a growing or grown culture, and then resealably reattached to the base.
With the increased emphasis on the efficacy of medical and research products, a need exists for an improved apparatus for effectively and efficiently carrying out biological culture production. The improved apparatus would better protect the person carrying out the procedure and would be simple and inexpensive to manufacture as compared to currently available devices.
Another desired feature for a microbiological assembly is the ability of the assembly to preserve a biological culture for long-term examination of the culture or for use as a reference or control. Cultures are sometimes preserved by being sealed within an anaerobic environment free from contamination and drying. For example, a permanent coverslip may be adhered or otherwise attached to a slide having a culture grown thereon with the coverslip covering the culture. A permanent adhesive may be used to seal the culture between the slide and coverslip. A coverslip may be mounted over a specimen on a slide by bonding the coverslip to the slide by means of a cement adhesive, natural or synthetic resin, or a photosensitive material such as an acrylate that solidifies under UV light.
In assemblies comprising removable compartments or covers, long-term preservation of a culture grown on an assembly support would heretofore require removing the compartment or cover, applying a permanent adhesive to either the assembly support or a coverslip, and permanently adhering the coverslip to the support with the culture therebetween. Great care must be taken so as not to contaminate the culture with the adhesive and so as to completely seal the culture from all sides. Furthermore, the permanent adhesive makes it difficult, if not impossible, to later remove the coverslip and gain future access to the culture. A need exists for a simpler and safer way to seal a culture for long-term preservation, examination, study and reference.
It would be desirable to provide a microbiological assembly which can be converted from a culture growth assembly to a microscopic examination assembly without the need to perform a manipulative step of applying an adhesive for a coverslip after a culture-growth cover is removed.